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3.1 INTRODUCTION

A number of mammalian species exhibit sex differences which are present prior to gonadal differentiation and are therefore not a consequence of gonadal hormone action. For example, striking sexual dimorphism is seen in the marsupials; both the scrotum and mammary glands are recognisable in the Tammar wallaby prior to gonadal differentiation (Shaw et a l, 1990).

More subtle pregonadal sex differences are seen among eutherians. These are manifest as a difference in developmental progression - at the same gestational age, males are developmentally advanced with respect to females. This advantage has been reported as a weight difference among 12.5 àpc rat fetuses (Scott and Holson, 1977), a difference in somite number among 8.5 dpc mice (Seller and Perkins-Cole, 1987) and a difference in head size and body length in first trimester human fetuses (Pedersen, 1980). It is also apparent in the significant XX-XY weight difference in mice at 10.5 6pc in Chapter 2, and in the more extensive data of Burgoyne et a/.(1995).

In Chapter 2, X^^'^0 mothers were mated to X^^-^Y fathers, primarily to enable within litter comparisons of X^O and X^O fetuses. A surprising result was the finding, in Experiment 2, that X^O fetuses, in addition to being larger than X^O fetuses, were also significantly larger than XX fetuses. The X^O-XX difference also bordered on significance in Experiment 1.

These results suggested that the XX-XY difiference seen at 10.5 àpc may, at least in part, be due to an effect of the difference in X-chromosomal constitution. This was unexpected because it has been assumed that the XX-XY differences seen in preimplantation and postimplantation stages have the same genetic basis, and the preimplantation difference has already been shown to be due to an accelerating effect of the Y-chromosome (Burgoyne, 1993). At the very least, the preimplantation Y-effect would be expected to be ‘carried-over’ into the postimplantation period. Yet only in Experiment 2a was there a suggestion of a Y-chromosomal effect, in so far as the X^O- XY comparison bordered on significance (0.1>P>0.05) with a one-tailed test.

Unfortunately, the x cross, although essential for the X^O-X^O comparison, is not suited to the detection of smaller differences between genotypes. This is due to the fact that with four genotypes being produced within small litters (the litter size of XO mothers may be as little as one half that of XX mothers - Morris, 1968), the standard error for the within litter comparisons is inevitably large (compare the SEMs in Tables 22a-c with those in Table 2.1). Indeed, the mean weighted difference for the X^O-XY comparison in Table 2.2a (0.060) is comparable in size to the significant differences reported in other chapters but fails to reach significance because of the large standard error. It was therefore decided to collect additional data on X^O-XY and X^O-XX differences using x X^^'^Y crosses since, in this case, only three genotypes (XX, XY and X^O) are produced and the litter size should be increased.

3.2 MATERIALS AND METHODS

3.2.1 Mice

The mice used for this experiment were derived from the same Patchy-fur stocks described in the previous chapter. females, homozygous for one PGK-1 isozyme, were mated to X^"^Y studs hemizygous for the alternative PGK-1 isozyme. Reciprocal crosses were set up so that the X^O-XX and X^O-XY comparisons would be equivalent to those from Experiments 2a and 2b in Chapter 2. The Y-chromosome present in the P af stock is known to be C3H in origin, since Paf arose in a subline of the C3H strain (Lane and Davisson, 1990). The offspring from these crosses have XX, X^O or XY genotypes. Note that the XO with a paternally derived X-chromosome is not seen amongst the offspring from these crosses (Figures 3.1a and 3.16).

Measurement of fetal weights, identification of genotypes and statistical analyses of data were as described in Chapter 2.

Figure 3.1a Cross used to generate X^O, XX and XY ofTspring within litters using XX mothers PI ■^^Pqf -^^Pqf X FI ■ ^aP af

o

Figure 3.16 Reciprocal cross used to generate X^O, XX and XY offspring within litters using XX mothers

PI ^oP<^ '^çoPaf '^Pctf's^ FI ■ ^^P qf o Y ■ ^^P af ■ ^^^af-^^P qf 'i^^aPqf'Y^ LEGEND: a = Pgk-I\ b = Pgk^P; + = wild type,

Paf=Patchy-fur mutation

3.3 RESULTS

The pooled data obtained from crosses 2a and 2b are presented in Table 2.\a. In agreement with the results presented in the previous chapter, the XY fetuses are significantly larger than XX fetuses; they are now also significantly larger than their X^O litter mates. The X^O fetuses are larger than XX litter mates and the difference approaches significance. The data from Table 3.la are illustrated in histogram form (Figure 3.2) as deviations from the ‘litter means’ against number of fetuses. The litter mean, against which deviations are calculated, is the average of the XX and XY litter means (since these genotypes occur with a greater frequency than the X^O genotype in this study). For the X^O genotype, the deviations are approximately equally distributed about the mean. In contrast, XX deviations are skewed significantly below litter means while XY deviations are skewed significantly above litter means, clearly demonstrating that XY fetuses are larger than X^O sibs, which are in turn larger than XX sibs.

Since these three genotypes were also compared in Experiments 2a and 2b, these data were pooled with the present data which considerably strengthens the comparisons between genotypes (see Table 3.16). It is clear from these combined data that XY fetuses are significantly larger than X^O fetuses, and that X^O fetuses are significantly larger than XX fetuses.

Table 3.1a Weight comparisons of X^O, XX and XY fetuses at 10.5 Ape

(pooled data from XX mothers)

Genotypes compared

No. of fetuses*

Mean+SE log weight** (wt in mg)*' Mean±SE weighted difference** Significance (P) XY 90 0.95 ±0.04 (9.0) 0.091 ±0.018 <0.0005^ XX 76 0.86 ±0.03 (7.3) x “ o 41 0.90 ±0.04 (8.0) 0.032 ±0.024 0.1-0.05* XX 59 0.86 ±0.04 (7.2) XY 74 0.96 ±0.04 (9.0) 0.064 ± 0.020 0.001-0.0005^ X ^ 41 0.90 ±0.04 (8.0)

"lumbers per genotype dififer between comparisons (not all litters contain all 3 genotypes) Vleans of litter means.

‘'Antilog of mean weight.

‘^Standard error calculated using the variance within genotypes within litters. * Borderline significance (2-tailed test)

Table 3.16 Weight comparisons of X^O, XX and XY fetuses at 10.5 dpc (pooled data from XO and XX mothers)

Genotypes compared

No. of fetuses®

Mean+SE log weight** (wt in mg)*^ Mean+SE weighted difference** Significance (P) XY 166 0.92 + 0.02 (8.3) 0.076 + 0.013 <0.0005^ XX 142 0.84 + 0.02 (7.0) x " o 72 0.90 + 0.02 (7.9) 0.044 + 0.017 0.02-0.01^ XX 116 0.85 + 0.02 (7.1) XY 135 0.93+0.02 (8.5) 0.047 + 0.016 0.0025-0.001 74 0.89 + 0.02 (7.7)

a,b,c,d, t as for Table 3. la

Figure 3.2 Histograms illustrating weight differences between XX, X^O and XY fetuses at 10.5 Ape (pooled data from XX mothers)

Log fetal weight data for 10.5 dpc fetuses are plotted as individual deviations from the average of the XX and XY ‘litter means’, i.e.

X =

N = number of fetuses

W = log fetal weight (deviations)

(/i) Figures in parentheses indicate the number of deviations falling above or below the litter mean

XX

XY

N X 10 5 - 10- 5 _ 10 5 - (28) (24) -feè‘‘î (28) (6 2 ) (20)

3.4 DISCUSSION

The postimplantation XX-XY difference has X and Y-chromosomal components

It has previously been assumed that the more advanced development of XY fetuses over XX fetuses has the same genetic basis in both the preimplantation and postimplantation periods. At preimplantation stages, this difference has been formally shown to be due to a Y-chromosome effect (Burgoyne, 1993). However, it is clear from the present results that the XX-XY postimplantation difference can now be resolved into two components - a Y-effect (XY^^^ fetuses are significantly larger than X^O sibs) and an effect of the difference in X-chromosome complement (X versus XX) which approaches significance in the pooled data from mothers (Table 3.1a) and is significant after combining data from and X^^’^O mothers (Table 3.16). In support of this the sum of the mean weighted differences, for the X^O-XX and X^O-XY comparisons in Table 3.1a, roughly approximates to the mean weighted XX-XY difference; while in Table 3.16, the mean weighted XX-XY difference is somewhat smaller than the sum of the two component comparisons.

While these observations underline the idea of two components contributing to the XX- XY difference, it is clear that the two components which contribute to the XX-XY difference are not quite additive. It must be emphasised that mean weighted differences can only be summed in this way when the times of processing and genetic backgrounds are comparable.

Further evidence for the existence of separate X and Y-chromosomal effects comes from crosses involving a Y-chromosome of RIII origin. A preimplantation XX-XY difference is not seen with the Y™ chromosome (Burgoyne, 1993; see also Chapter 5) but a clear XX-XY™ difference is seen at 10.5 6pc. Furthermore, X^O and XY™ fetuses are equivalent in size at 10.5 àpc (Burgoyne et a/., 1995). The XX-XY™ difference is therefore entirely explained by the difference in X-chromosome constitution, since the Y™ chromosome confers no developmental advantage at preimplantation stages or later

In summary, the XX-XY difiference, measured at 10.5 àpc, comprises two components: an accelerating efifect of the Y-chromosome which is ‘carried-over’ fi'om the preimplantation period and an X-chromosome efifect.

The origin o f X X developmental retardation

What aspect of the difiference in X-chromosome constitution is responsible for the slight retardation of XX fetuses as compared to X^O and XY fetuses? In view of the retarding efifect of the paternally imprinted X on X^O development (Chapter 2), it is tempting to attribute the retardation of XX fetuses solely to the presence of a paternally derived X. However, in the first tissues in which X-inactivation occurs (in the trophectoderm at 3.5<^c and primary endoderm at 4.5dpc) it is the paternal X which is preferentially inactivated (Takagi and Sasaki, 1975; West et al, 1977; Harper et al, 1982), so these tissues should be equivalent in XX and XY embryos, in each case having a single X^ chromosome expressed.

Once random X-inactivation has occurred in the epiblast {ca. 6.5àpc), XX embryos will dififer fi'om XY embryos in having approximately half of their cells, on average, expressing a paternal rather than a matemal X, but the very fact that X-inactivation is random at this time has been taken as evidence that any differential X imprint between the paternal and matemal X-chromosomes has been erased (Lyon and Rastan, 1984). If this is so, then there is once again no basis for a reduction in the level of X-chromosome expression in XX compared to XY embryos.

However, it is possible that these two effects of X-chromosome imprinting (on the choice of X to be inactivated in early differentiating tissues and on early embryonic growth) are mediated through different X-chromosome regions or different forms of imprint. If this were the case, one would have to assume differential transcriptional activity fi'om the matemal and patemal X-chromosomes following X-inactivation in the embryonic lineages. Thus XX females would have a level of X-chromosome transcription intermediate between X^O or X^Y fetuses and X^O fetuses.

Inappropriate X-chromosomal dosage

Rather than a reduction in X-chromosome activity in XX embryos being responsible for the XX-XY difiference, it is possible that, prior to X-inactivation, tissues vAth both X- chromosomes active have too much X-activity. Takagi and Abe (1990) have presented compelling evidence that a surfeit of X-chromosome activity, due to a failure of X- inactivation, is highly deleterious for the early mouse embryo. It is therefore quite possible that expression from two X-chromosomes prior to X-inactivation is suboptimal for normal development.

Studies of sex chromosome trisomy indicate that an additional matemal X is more deleterious than an additional patemal X in mice (Shao and Takagi, 1990; Takagi, 1991) and can result in severe growth retardation or abnormal development characterised by deficient extraembryonic structures as early as 6.5 àpc (Tada et al., 1993). The harmfiil effects of two X^ chromosomes may be due to the imprinted resistance of matemal X-chromosomes to inactivation (Lyon and Rastan, 1984), a normal requirement in the extraembryonic membranes (Shao and Takagi, 1990).

These observations are supported by the finding that XO parthenogenotes, while still inviable, are less severely affected than XX parthenogenotes (Mann and Lovell-Badge, 1988). Unfortunately, in these experiments it is difficult to assess the specific effects of the X- chromosome because of the generally detrimental, and ultimately lethal, effects due to the uniparental inheritance of a complete set of autosomes (McGrath and Solter, 1984).

In view of these findings, one would predict that mice trisomie for the X-chromosome would be inviable. Indeed, very few cases of surviving 41,XXX mice have been reported (Endo and Watanabe, 1989; Matsuda and Chapman, 1992; Sakurada et al., 1994) but in none of these cases was the supemumerary X-chromosome confirmed as matemal in origin. XXX mice generated using the Rb(X.2)2Ad Robertsonian translocation system (Adler et al., 1989) which inherit two matemally derived X-chromosomes die at mid-gestation (Tada et al.,

result in a postimplantation 0-XX developmental difference and has a non-significant XX- XY postimplantation difference (Omoe and Endo, 1993).

In contrast to the situation in the mouse, two matemal X-chromosomes are not detrimental to development in humans. Molecular analysis has shown that amongst triple X (47,XXX) females, the extra X-chromosome is of matemal origin in 90 per cent of cases and these individuals show no striking somatic abnormalities (May et al, 1990). Similarly, 47,X^X^Y Klinefelter males are seen in nearly 50 per cent of surviving XXY conceptions (Jacobs et al,

1988). Although some selection operates against both XXX and XXY conceptions in utero, there is no evidence of preferential selection against individuals with two maternal X- chromosomes. Neither are there any striking phenotypic effects associated with the parental origin of the extra X-chromosome.

Further evidence against a deleterious effect of two matemal X-chromosomes in humans comes from the observation that large duplications of regions of the X-chromosome (which do not undergo X-inactivation) seen in some male patients do not appear to be lethal (Schmidt a/., 1991).

In this chapter it has been argued that the XX-XY difference observed at 10.5 àpc is due to two separate genetic components: an X-chromosome dosage effect (presumably operating prior to X-inactivation) and a Y-chromosomal effect ‘carried-over’ from preimplantation stages. On the basis of this explanation, predictions can be made about effects on the developmental progression of fetuses with additional sex chromosomes. Thus, in terms of developmental progress, XXY fetuses are expected to be intermediate between XX and XY individuals, while XYY fetuses might be expected to be ahead of XY fetuses. The next chapter uses a breeding system which generates ‘XXY’ females and XYY’ males among the offspring, in order to test these predictions. The additional Y-chromosome is unusual in that it has a deletion for the Sry gene. This means that the XX versus XXY’ female comparison also tests the suggestion made by Zwingman et a/. (1993) that Sry may be responsible (at least in part) for the accelerating effect of the Y-chromosome.

CHAPTER 4

EFFECTS OF AN ADDITIONAL SRY-NEGATIVE